Syntheses of mycobactin analogs as potent and selective inhibitors of Mycobacterium tuberculosis

Article abstract of DOI:10.1039/c2ob26077h

Three analogs of mycobactin T, the siderophore secreted by Mycobacterium tuberculosis (Mtb) were synthesized and screened for their antibiotic activity against Mtb H₃₇Rv and a broad panel of Gram-positive and Gram-negative bacteria. The synthetic mycobactins were potent (MIC₉₀ 0.02-0.88 μM in 7H12 media) and selective Mtb inhibitors, with no inhibitory activity observed against any other of the microorganisms tested. The maleimide-containing analog 40 represents a versatile platform for the development of mycobactin-drug conjugates, as well as other applications.

Full text of DOI:10.1039/c2ob26077h

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PAPER
Syntheses of mycobactin analogs as potent and selective inhibitors of
Mycobacterium tuberculosis†
Raúl E. Juárez-Hernández,a Scott G. Franzblaub and Marvin J. Miller*a
Received 4th June 2012, Accepted 2nd August 2012
DOI: 10.1039/c2ob26077h
Three analogs of mycobactin T, the siderophore secreted by Mycobacterium tuberculosis (Mtb) were
synthesized and screened for their antibiotic activity against Mtb H37Rv and a broad panel of Gram-
positive and Gram-negative bacteria. The synthetic mycobactins were potent (MIC90 0.02–0.88 μM in
7H12 media) and selective Mtb inhibitors, with no inhibitory activity observed against any other of the
microorganisms tested. The maleimide-containing analog 40 represents a versatile platform for the
development of mycobactin-drug conjugates, as well as other applications.
Introduction
Tuberculosis (TB), the disease caused by Mycobacterium tuber-
culosis (Mtb), is one of the most critical infections burdening the
world. It has been estimated that in 2009 alone, TB-related mor-
tality reached 1.7 million people (1.3 million, being HIV-nega-
tive; 0.4 million HIV-positive). Furthermore, about one third of
the planet’s population is currently infected.1 The complexity
and length of TB treatment has led to poor patient compliance,
and, together with the emergence of multi-drug resistant (MDR)
and extensively-drug resistant (XDR) strains,2 the urgency for
the development of new antibiotic agents has increased.
We are interested in exploiting the process for iron assimilation
by Mtb to deliver compounds of interest inside the cell under the
so-called “Trojan Horse” approach.3 Almost all organisms require
iron, an essential element in many metabolic processes. Ensuring
Fig. 1 General structures of the Mtb siderophores.9
its acquisition is therefore, a high priority for any pathogen during
the course of an infectious cycle.4 Although highly abundant,
ferric iron forms insoluble salts in the presence of oxygen and
water, rendering the effective concentration of the metal in
Gobin in 19959,10 (Fig. 1). A series of studies revealed the
aqueous solutions (10−9–10−10 M, pH = 7),5 lower than the
importance of mycobactin T as a growth factor and agent of
optimal levels for bacterial growth (10−6–10−7 M). In biological
virulence.11
systems, these levels are further decreased (10−15–10−25 M) due
During the characterization of these molecules, Snow
to sequestration by molecules such as transferrin and lactoferrin.6
observed that the siderophore produced by one species of myco-
The iron uptake system of Mtb utilizes two, structurally
bacteria had an inhibitory effect on the growth of a different
strain if added externally. Demonstrating this hypothesis would
prove challenging because of the isolation and complexity of
related, high affinity, Fe3+-chelators: mycobactin T, isolated by
Snow in 1965,7,8 and the carboxymycobactin T, isolated by
these compounds.
In 1983, our group reported the total synthesis of mycobactin
aDepartment of Chemistry and Biochemistry, University of Notre Dame,
251 Nieuwland Science Hall, Notre Dame, IN 46556, USA.
S2 (1, Fig. 2), the first example of an iron-binding analog that
was found to be inactive as a growth inhibitor of Mtb.12 The
E-mail: mmiller1@nd.edu
bInstitute for Tuberculosis Research, College of Pharmacy, University of
assembly of this molecule emphasized the importance of the
Illinois at Chicago, Chicago, IL 60612, USA
long acyl chain present in the natural mycobacterial sidero-
†Electronic supplementary information (ESI) available: Experimental
1
procedures of chemistry and microbiology, H and 13C NMR spectra of
synthesized compounds. See DOI: 10.1039/c2ob26077h
phores, which appeared to be critical for adequate activity.
Although significant work is needed to completely understand
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Fig. 2 Synthetic and structurally-related analogs of mycobactin T.
the process of iron-uptake in Mtb,13–18 it is hypothesized that
mycobactin T effectively diffuses through the cell membrane,
where it is involved in iron acquisition, whether by exchange
with the excreted carboxymycobactins9,10 or through sequestra-
tion from the iron pools within the macrophage.13,14 Considering
this mode of action, the advantage of a long, hydrophobic acyl
substituent, became more evident, which was corroborated with
the assembly of mycobactin S (2, Fig. 2) in 1997, when the syn-
thetic analog was shown to effectively inhibit (>99%) Mtb
structural requirements needed for Mtb activity: oxazoline
moiety, stereochemistry and substitution near the ester bond, and
a long acyl chain.
Our group also reported the synthesis of a mycobactin-artemi-
sinin conjugate in 2011,23 the first approach towards the develop-
ment of such Trojan Horse molecules using a mycobacterial
siderophore. Because the naturally occurring mycobactins do not
possess a site for chemical elaboration, the core structure was
modified to include a diaminopropionate spacer, with a N-Boc
protected functionality (9, Fig. 2) that allowed derivatization.
Interestingly, this precursor, itself, was shown to have potent
inhibitory activity against Mtb H37Rv (MIC = <0.2 μg mL−1).
The drug-conjugate (10, Fig. 2) had strong and specific inhi-
bition of Mtb H37Rv (MIC = 0.39 μg mL−1), MDR Mtb (MIC =
0.16–1.25 μg mL−1), XDR Mtb (MIC = 0.078–0.625 μg mL−1),
H37Rv at a concentration of 12.5 μg mL−1 19
.
Further structural modification of the mycobactin core was
reported in 2007,20 with the substitution of the oxazoline moiety
by a catechol group and the syntheses of two analogs (4–5,
Fig. 2) that were hypothesized to be effective growth promoters
for mycobacteria. The artificial siderophores were tested in a
chrome azurol S (CAS) assay21 to confirm their iron binding
capabilities, and screened for antibacterial activity against a
panel of Gram-positive, and Gram-negative bacteria, as well as
mycobacteria. While no antibiotic activity was observed, inclu-
ding Mtb which was not inhibited at 6.25 μg mL−1 (via the
Tuberculosis Antimicrobial Acquisition and Coordinating Faci-
lity, www.taacf.org), both analogs had almost identical growth
promoting effects as that of the naturally occurring mycobactin
J,9,13,15 particularly with strains of M. smegmatis which were
expected to be more receptive towards these siderophores.
In 2008, Fennell reported the syntheses of three analogs of
amamistatin B, a natural product isolated from the actinomycete
Nocardia asteroides, and screened them against MCF-7 and
PC-3 human tumor cells. Because of their structural similarity
with the mycobactins, these analogs were screened against Mtb
but only analog 6 (Fig. 2) was shown to have modest inhibitory
activity (MIC = 47 μM),22 perhaps stressing the importance of
and against four strains of Plasmodium falciparum (IC50
=
0.004–0.005 μg mL−1), the causative agent of malaria in
humans. Because no inhibitory activity was observed at the
highest concentration tested (2 mM) against a broad panel of
bacteria, this synthetic analog represents a clear example of the
ability to develop disease-specific agents by exploiting the iron-
uptake as a biological target.
One of the main difficulties in the syntheses of drug-conju-
gates is the tailoring of the chemistry needed to couple the drug
of interest with the siderophore of choice, which can result in
extensive exploration and protective group strategies to find the
adequate conditions. With the objective of developing a more
universal platform towards mycobactin-conjugates, as well as
other applications, we decided to synthesize a maleimide-con-
taining analog (40, Fig. 3), while maintaining the rest of the
mycobactin T structure unaltered. Herein, we describe the syn-
theses of new mycobactin analogs suitable for eventual use in
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91% yield. Removal of the phthalimide protecting group with
methylhydrazine, gave the desired aniline 18 in 75% yield.
Aniline 18 was deprotected by hydrogenolysis to afford frag-
ment 20, and because the synthesis of mycobactin 36, vide infra,
required a re-protection strategy, intermediate 18 was reacted
with di-tert-butyl dicarbonate to afford 19 in 57% yield, which
upon hydrogenolysis gave oxazoline 21 in quantitative yield.
Incorporation of the N-Boc protecting group at the beginning of
the synthetic route proved to be less efficient and problematic.
Synthesis of Cobactin T
Fig. 3 Retrosynthetic approach for the assembly of maleimide-contain-
ing mycobactin (T) analog 40.
As described earlier, intermediate 22,22 was deprotected using
HBr in acetic acid to afford the corresponding salt 23
(Scheme 2), which was then coupled to carboxylic acid 2526 to
afford cobactin T 26,27 in 62% yield.
preparing novel drug conjugates and report that the new con-
structs themselves have inherent anti-TB activity.
Mycobactin assembly
Results and discussion
Protected linear lysine based hydroxamate 27,23 was hydrolyzed
using LiOH to afford carboxylic acid 28 (Scheme 3) in quantita-
tive yield. EDC·HCl-mediated coupling of this intermediate with
cobactin 26 provided the right-hand fragment of the mycobactin
T core in 88% yield. Deprotection with HBr in acetic acid
afforded the corresponding salt 30, which was then coupled with
oxazoline 20 to provide O-Bn protected intermediate 31 in 42%
yield, while coupling of 30 with oxazoline 21 provided 32 in
52% yield.
After the appropriate conditions for the removal of the O-Bn
protecting groups were determined, intermediate 31 was sub-
jected to hydrogenolysis in THF under atmospheric pressure to
afford 33 (Scheme 3), which was immediately re-protected, to
facilitate purification, using acetic anhydride to afford the desired
Syntheses of oxazoline components
Commercially available 4-aminosalicylic acid was esterified
under acidic conditions to afford intermediate 1124 (Scheme 1),
which was then refluxed with phthalic anhydride in acetic acid.
The phenol group of 12,25 was protected using benzyl bromide
and K2CO3 to afford fully protected intermediate 13 in 79%
yield. Saponification of the methyl ester with KOH also effected
ring opening of the phthalimide moiety. Amide 16 was then
obtained in 77% yield from a two-step sequence by reacting
intermediate 14 with oxalyl chloride (which induced phthalimide
ring closure), followed by coupling with L-serine-OBn·HCl 15.
Oxazoline cyclization was performed using DAST (diethyl-
aminosulfur trifluoride) to afford the desired intermediate 17 in
Scheme 1 Syntheses of oxazoline fragments 20 and 21.
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Scheme 2 Assembly of Cobactin T 26.
Scheme 3 Elaboration of mycobactin analogs 34, 36 and 40.
mycobactin analog 34 in 70% yield. The acetylation of the
poorly nucleophilic aniline prompted the synthesis of N-Boc-
protected oxazoline 21, which was elaborated to intermediate 32.
After hydrogenolysis and acetylation, the desired mycobactin 36
was isolated in 87% yield.
esters of the oxazoline component possess moderate growth
inhibitory activity against Mtb.30 Therefore, oxazoline intermedi-
ates 17, 18, and 19 were tested against Mtb H37Rv (Table 1);
however, only minimal activity was observed with analog 19
(7H12: MIC = 23.57 μM; GAS: MIC = 5.81 μM). Mycobactin
analogs 34 and 36 showed potent inhibitory activity (7H12: MIC
= 0.02–0.09 μM), while 40 displayed potent but slightly
decreased activity (7H12: MIC = 0.88 μM). Antibiotic activity
against non-replicating Mtb, was determined through the Low-
Oxygen-Recovery Assay (LORA)31 where the mycobactin
analogs were found to be inactive (MIC > 50 μM, Table S2,
Maleimide-containing mycobactin
Treatment of 36 with TFA cleanly removed the N-Boc group to
give 37 (Scheme 3). Maleimide-containing carboxylic acid 38
was prepared according to the literature,28 and reacted with
oxalyl chloride to afford the corresponding acid chloride 39,
which was then used to acylate 37. After purification, the desired
mycobactin analog 40 (Fig. 3) was obtained in 89% yield.
ESI†). Interestingly, the synthetic siderophores 34 (IC50
=
21.50 μM) and 36 (IC50 = 22.37 μM) displayed greater cyto-
toxicity against Vero cells32 than the maleimide analog 40
(IC50 = >50 μM).
These compounds demonstrated little to no inhibition of a
broad panel of Gram-positive and Gram-negative bacteria
(see Table S1, ESI†).33,34 The fact that limited antibiotic activity
can be observed for mycobactin 40 (diameters of growth
inhibition 13–18 mm), might be attributed to the maleimide-
linker, a Michael-acceptor, though as indicated above, the same
Biological activity
Based on our previous synthetic efforts related to the syntheses
of mycobactin analogs, we can conclude that other than the fully
elaborated, O-Bn deprotected siderophores, the hydrophobic
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Table 1 Antibacterial activity of compounds in the Microplate Alamar
acetic acid was refluxed at 118 °C (bath temperature) for 22 h
under argon. The resulting solution was allowed to cool to room
temperature and poured over ice. Upon thawing, the newly
formed precipitate was filtered, air dried and rinsed with cold
(0 °C) diethyl ether (20 mL), air dried again and purified
by silica gel chromatography using CH2Cl2 as the eluent (TLC,
Rf = 0.29) to afford 7.79 g (87%) of 12 as white crystals: mp
Blue Assay (MABA)29
MIC90 (μM) determined in:
Compound
7H12a
GASb
17
18
19
34
36
40
>128c
>128c
23.57
0.09
0.02
0.88
>128
60.97
5.81
0.43
2.88
1.02
1
208 − 210 °C; H NMR (600 MHz, CDCl3) δ 10.89 (s, 1H),
8.00–7.96 (m, 3H), 7.83 (dd, J = 5.6, 2.9 Hz, 2H), 7.19 (d, J =
1.8 Hz, 1H), 7.07 (dd, J = 8.5, 2.1 Hz, 1H), 3.99 (s, 3H);
13C NMR (150 MHz, CDCl3) δ 170.2, 166.8, 162.2, 138.3,
134.9, 131.7, 130.7, 124.2, 117.0, 115.3, 111.8, 52.7; HRMS
a 7H12 = 7H9 broth base media with BSA, casein hydrolysate, catalase,
palmitic acid. b GAS = glycerol-alanine-salts media. c GAST = iron-
deficient GAS media with added Tween 80.11 Values reported are the
average of three individual measurements.
(ESI) m/z [M + Na]+: calcd for C16H11NNaO5 , 320.0529;
+
found, 320.0558; HRMS (ESI) m/z [M + H]+: calcd for
+
C16H12NO5 , 298.0710; found, 298.0722.
maleimide derivative was not cytotoxic. It is also important to
highlight that when testing the fully elaborated mycobactins, a
significant level of precipitation was observed during the assay,
probably due to hydrophobicity of these compounds, an effect
also observed when performing chrome azurol S (CAS) assays,
to determine the metal-binding properties of the siderophores.20
Benzyl ether, 13
To a suspension of 5 g (16.8 mmol) of phenol 12, in 44 mL of
anhydrous DMF under argon, was added 5.87 g (42.4 mmol)
of K2CO3 in one portion, followed by 5.40 mL (45.1 mmol) of
benzyl bromide. The resulting mixture was stirred under argon
for 24 h, before being concentrated under vacuum. The residue
was dissolved in CH2Cl2 (30 mL) and washed with H2O
(30 mL). The aqueous layer was further extracted with CH2Cl2
(4 × 15 mL). The organic extracts were combined and washed
with brine (2 × 50 mL), dried over Na2SO4, filtered, concen-
trated and the residue was re-crystallized from CH2Cl2/hexanes
to afford 5.19 g (79%) of 13 as a crystalline, off-white solid:
1H NMR (600 MHz, CDCl3) δ 8.01–7.96 (m, 3H), 7.83 (dd, J =
5.4, 3.1 Hz, 2H), 7.54–7.51 (m, 2H), 7.43–7.39 (m, 2H),
7.35–7.31 (m, 1H), 7.26 (d, J = 1.8 Hz, 1H), 7.20 (dd, J = 8.4,
1.9 Hz, 1H), 5.22 (s, 2H), 3.93 (s, 3H); 13C NMR (150 MHz,
CDCl3) δ 167.0, 166.2, 158.8, 136.5, 136.5, 134.9, 132.6, 131.7,
128.8, 128.1, 127.1, 124.1, 119.9, 118.1, 111.7, 71.0, 52.3;
Conclusions
We have synthesized and tested three mycobactin (T) analogs
while pursuing the development of maleimide-containing myco-
bactin 40. This molecule represents a versatile platform for the
elaboration of siderophore-drug conjugates via a thiol-maleimide
reactive system. The proposed approach can simplify the syn-
thetic route by reducing the use of protecting groups and provid-
ing a more convergent sequence. In comparison with the
assembly of mycobactin analog 9, used in the synthesis of arte-
misinin conjugate 10,23 we have minimized the changes in the
structural identity of mycobactin T, which might be of impor-
tance for biological recognition. The coupling with antibiotics or
other molecules of interest through a unique strategy may allow
rapid access to conjugates, while the potentially reversible thiol
addition and the use of different thiol linkers could facilitate
drug delivery. Our synthetic siderophores were demonstrated to
HRMS (ESI) m/z [M + Na]+: calcd for C23H17NNaO5 ,
410.0999; found, 410.0987.
+
Dicarboxylic acid, 14
be potent and selective inhibitors against Mtb H37Rv (MIC90
=
To a suspension of 0.98 g (2.53 mmol) of methyl ester 13 in
25 mL of MeOH was added 0.92 g (16.4 mmol) of anhydrous
KOH in one portion, followed by 1 mL of H2O. The mixture
was allowed to stir at room temperature. After 18.5 h, the reac-
tion was judged complete by TLC, dicarboxylic acid Rf = 0.28
(1 : 9, MeOH/CH2Cl2). The solution was concentrated under
vacuum to afford a colorless oil that was dissolved in 25 mL of
H2O, and acidified to pH = 2 with 1 M HCl. The precipitate that
formed was separated by filtration and used without further
0.02–0.88 μM in 7H12 media, MIC90 = 0.43–2.88 μM in GAS
media), while displaying no inhibitory activity against a broad
panel of bacteria, underlining the complexities of the iron-uptake
system in Mycobacterium tuberculosis. While these mycobactins
might have an inhibitory effect by interfering with the iron
trafficking of Mtb, much is still to be learned about the exact
process of iron delivery within the cell, and the role of the struc-
tural differences between the mycobactin analogs (natural and
synthetic) and the observed biological activity. Use of the malei-
mide derivative for drug and bioconjugation studies will be
reported in due course.
1
purification: H NMR (600 MHz, CD3OD) δ 8.04 (dd, J = 7.9,
0.9 Hz, 1H), 7.86 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 1.8 Hz, 1H),
7.69–7.65 (m, 1H), 7.59 (td, J = 7.6, 1.2 Hz, 1H), 7.56–7.53 (m,
3H), 7.40–7.36 (m, 2H), 7.33–7.29 (m, 1H), 7.21 (dd, J = 8.5,
2.1 Hz, 1H), 5.25 (s, 2H); 13C NMR (150 MHz, CD3OD) δ
171.6, 169.2, 169.2, 160.6, 145.9, 140.3, 138.0, 134.1, 133.5,
131.6, 131.0, 130.6, 129.7, 129.2, 128.9, 128.7, 116.5, 113.1,
Experimental
N-Phthalimide-protected aminosalicylate, 1225
A suspension of 5.01 g (29.9 mmol) of methyl ester 11,24 and
5.55 g (37.5 mmol) of phthalic anhydride in 62 mL of glacial
106.4, 72.0; HRMS (ESI) m/z [M +
Na]+: calcd for
+
C22H17NNaO6 , 414.0948; found, 414.0959.
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Salicylserine derivative, 16
(600 MHz, CDCl3) δ 7.99–7.95 (m, 3H), 7.82 (dd, J = 5.4,
3.1 Hz, 2H), 7.54–7.52 (m, 2H), 7.42–7.28 (m, 8H), 7.24 (d, J =
1.8 Hz, 1H), 7.21–7.19 (m, 1H), 5.32–5.20 (m, 4H), 5.04 (dd,
J = 10.6, 7.9 Hz, 1H), 4.70–4.66 (m, 1H), 4.59 (dd, J = 10.6,
8.5 Hz, 1H); 13C NMR (150 MHz, CDCl3) δ 171.2, 167.0,
165.2, 158.3, 136.6, 135.8, 135.7, 134.9, 132.3, 131.7, 128.8,
128.7, 128.6, 128.5, 127.9, 127.1, 124.1, 118.4, 116.7, 111.8,
71.1, 69.5, 69.1, 67.4; HRMS (ESI) m/z [M + H]+: calcd for
To a suspension of 1.13 g (2.89 mmol) of combined lots of
dicarboxylic acid 14 in 23 mL of anhydrous CH2Cl2 under
argon was added 1 mL (11.5 mmol) of oxalyl chloride, (COCl)2
slowly by syringe, followed by 4 drops of anhydrous DMF. The
reaction mixture bubbled profusely. The mixture was stirred
under argon for 3.5 h until judged complete by TLC, N-phthal-
imide acid chloride Rf = 0.23 (1 : 1, EtOAc/hexanes). The solu-
tion was concentrated under vacuum and sequentially co-
evaporated with toluene (3 × 10 mL) and CHCl3 (3 × 10 mL) to
afford an orange solid. The resulting acid chloride was dissolved
in 23 mL of anhydrous CH2Cl2 under argon and cooled to
−78 °C using a dry ice/acetone bath. Then 1.21 mL (6.95 mmol)
of anhydrous DIPEA was added slowly, followed by 0.71 g
(3.06 mmol) of L-serine-OBn·HCl 15, in one portion. The
mixture was allowed to warm to room temperature and stir under
argon overnight. After being judged complete by TLC, N-phtha-
limide amide Rf = 0.31 (1 : 1, ethyl acetate/hexanes), the crude
mixture was diluted with 30 mL of CH2Cl2 and poured over
50 mL of H2O, the layers were separated, and the organic extract
was washed with H2O (40 mL), 10% (w/v) citric acid (3 ×
40 mL), H2O (40 mL), satd. NaHCO3 (3 × 40 mL), H2O
(40 mL), and brine (3 × 40 mL), dried over Na2SO4, filtered and
concentrated. The residue was purified by silica gel chromato-
graphy using a gradient from 35 : 65 : 2 to 100 : 0 : 2 of EtOAc/
hexanes/isopropyl alcohol as the eluent to afford 1.23 g (77%
over two steps) of 16 as a white, flaky solid: mp 170 − 171 °C;
1H NMR (600 MHz, CDCl3) δ 8.75 (d, J = 7.3 Hz, 1H), 8.33 (d,
J = 8.5 Hz, 1H), 7.98 (dd, J = 5.4, 3.1 Hz, 2H), 7.83 (dd, J =
5.3, 2.9 Hz, 2H), 7.50–7.46 (m, 2H), 7.41–7.28 (m, 10H),
5.27–5.12 (m, 4H), 4.91–4.88 (m, 1H), 3.97–3.88 (m, 2H), 2.08
(t, J = 6.2 Hz, 1H); 13C NMR (150 MHz, CDCl3) δ 170.3,
167.0, 165.0, 157.3, 136.2, 135.5, 135.3, 134.9, 133.1, 131.7,
129.1, 129.0, 128.8, 128.6, 128.6, 128.3, 124.2, 120.6, 119.1,
110.8, 72.0, 67.5, 63.8, 55.6; HRMS (ESI) m/z [M + Na]+: calcd
+
C32H25N2O6 , 533.1707; found, 533.1708.
4-Amino oxazoline, 18
A solution of 1.63 g (3.1 mmol) of N-phthalimide protected oxa-
zoline 17 in a 60 mL of anhydrous THF was cooled to 0 °C (ice-
H2O bath) for 15 min, then 0.18 mL (3.4 mmol) of methylhydra-
zine was added dropwise and the solution was allowed to warm
to room temperature. After 3 h, the reaction was judged incom-
plete by TLC, aniline Rf = 0.54 (EtOAc), so it was again cooled
to 0 °C and 0.05 mL (0.75 mmol) of methylhydrazine was
added slowly. The reaction was allowed to warm to room temp-
erature and stirred for 2 h until completion. The solvent was
evaporated and CHCl3 was added to precipitated phthalimide
by-product, which was then removed by filtration. The filtrate
was concentrated and the residue was purified by silica gel
chromatography with 2 : 98 isopropyl alcohol/CH2Cl2 as the
1
eluent to afford 0.93 g (75%) of 18 as a light-tan oil: H NMR
(600 MHz, CDCl3) δ 7.69 (d, J = 8.5 Hz, 1H), 7.53–7.50 (m,
2H), 7.41–7.26 (m, 8H), 6.26–6.22 (m, 2H), 5.30–5.19 (m, 2H),
5.15–5.09 (m, 2H), 4.97 (dd, J = 10.6, 7.9 Hz, 1H), 4.62–4.57
(m, 1H), 4.51 (dd, J = 10.6, 8.5 Hz, 1H), 3.98 (br. s., 2H);
13C NMR (150 MHz, CDCl3) δ 171.7 165.9, 159.6, 151.2,
137.2, 135.8, 133.4, 128.7, 128.6, 128.5, 128.5, 127.6, 126.8,
107.4, 106.8, 100.1, 70.6, 68.9, 68.9, 67.2; HRMS (ESI) m/z
[M + Na]+: calcd for C24H22N2NaO4 , 425.1472; found,
+
+
425.1493. HRMS (ESI) m/z [M + H]+: calcd for C24H23N2O4 ,
+
for C32H26N2NaO7 , 573.1632; found, 573.1628; HRMS (ESI)
403.1652; found, 403.1667.
+
m/z [M + H]+: calcd for C32H27N2O7 , 551.1813; found,
551.1784.
N-Boc-Protected oxazoline, 19
N-Phthalimide-protected oxazoline, 17
To a solution of 0.937 g (2.32 mmol) of 4-amino oxazoline 18
in 10 mL of anhydrous THF under argon, was slowly added a
premixed solution of 1.45 g (6.61 mmol) of di-tert-butyl dicar-
bonate in 10 mL of anhydrous CH3CN. The reaction was stirred
at 60 °C for 68 h while periodically monitored by TLC. N-Boc-
protected oxazoline Rf = 0.28 (1 : 1, ethyl EtOAc/hexanes). The
reaction mixture was concentrated and purified by silica gel
chromatography using 4 : 6 ethyl EtOAc/hexanes to afford
0.664 g (57%) of 19 as an off-white foam/residue: 1H NMR
(600 MHz, CDCl3) δ 7.77 (d, J = 8.5 Hz, 1H), 7.56–7.53 (m,
2H), 7.42 (br. s., 1H), 7.40–7.26 (m, 8H), 6.78 (dd, J = 8.5,
2.1 Hz, 1H), 6.69 (s, 1H), 5.32–5.17 (m, 4H), 4.99 (dd, J = 10.4,
8.1 Hz, 1H), 4.63 (dd, J = 8.5, 7.9 Hz, 1H), 4.54 (dd, J = 10.6,
8.5 Hz, 1H), 1.53 (s, 9H); 13C NMR (150 MHz, CDCl3) δ
171.5, 165.5, 159.0, 152.4, 143.0, 137.0, 135.7, 132.6, 128.8,
128.6, 128.5, 128.5, 127.7, 127.1, 111.4, 110.1, 103.3, 81.3,
70.7, 69.2, 69.0, 67.4, 28.5; HRMS (ESI) m/z [M + H]+: calcd
A solution of 3.03 g (5.5 mmol) of combined lots of 16 in
110 mL of anhydrous CH2Cl2 under argon was cooled to
−78 °C in a dry-ice/acetone bath. Then 0.84 mL (6.4 mmol) of
diethylaminosulfur trifluoride (DAST) was added slowly and the
reaction was allowed to proceed under argon for 5 h, while being
monitored every 60 min by TLC. After the reaction was judged
complete by TLC Rf = 0.38 (1 : 1, EtOAc/hexanes), 2.05 g
(14.8 mmol) of anhydrous K2CO3 were added to the reaction in
one portion and the mixture was allowed to warm to room temp-
erature. The solution was poured over saturated aqueous
NaHCO3 (80 mL) and the layers were separated. The aqueous
layer was then extracted with CH2Cl2 (2 × 60 mL) and the
organic layers were combined and washed with saturated
NaHCO3 (2 × 60 mL), H2O (2 × 60 mL), brine (3 × 60 mL),
dried over Na2SO4, filtered and concentrated to afford an off-
white solid that was re-crystallized from CH2Cl2/EtOAc to
1
+
afford 2.67 g (91%) of 17 as a white cotton-like solid: H NMR
for C29H31N2O6 , 503.2177; found, 503.2165.
This journal is © The Royal Society of Chemistry 2012
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Protected caprolactam, HBr salt 23,22 and 0.188 g (1.39 mmol)
of HOBt in 12 mL of anhydrous CH3CN under argon and
0.24 mL (1.38 mmol) of anhydrous DIPEA were added drop-
wise, followed by addition of 0.293 g (1.52 mmol) of EDC·HCl
in one portion. The reaction was allowed to proceed for 25 h and
monitored by TLC, cobactin T Rf = 0.16 (EtOAc, CAM stain).
Then the reaction was partitioned between 25 mL of EtOAc and
25 mL of H2O. The aqueous layer was extracted with EtOAc
(4 × 15 mL), the organic extracts were combined, washed with
brine (2 × 30 mL), dried over Na2SO4, filtered, concentrated and
purified by silica gel chromatography with EtOAc as the eluent
to afford 0.249 g (62%) of 26 as a white solid: 1H NMR
(600 MHz, CDCl3) δ 7.45–7.36 (m, 5H), 7.02 (d, J = 6.2 Hz,
1H), 5.00 (d, J = 10.3 Hz, 1H), 4.91 (d, J = 10.3 Hz, 1H), 4.50
(ddd, J = 11.4, 6.5, 1.8 Hz, 1H), 4.24–4.15 (m, 1H), 3.89 (d, J =
2.9 Hz, 1H), 3.62 (dd, J = 16.1, 11.4 Hz, 1H), 3.53–3.48 (m,
1H), 2.44 (dd, J = 15.3, 2.6 Hz, 1H), 2.33 (dd, J = 15.3, 9.1 Hz,
1H), 2.04–1.98 (m, 1H), 1.96–1.90 (m, 1H), 1.77–1.64 (m, 2H),
1.54–1.37 (m, 2H), 1.24 (d, J = 6.2 Hz, 3H); 13C NMR
(150 MHz, CDCl3) δ 171.8, 170.2, 135.2, 129.9, 129.2, 128.9,
77.1, 65.0, 53.0, 51.9, 43.9, 31.6, 27.8, 26.4, 22.8; HRMS (ESI)
4-Amino oxazoline acid, 20
To a solution of 0.084 g (0.21 mmol) of 4-amino oxazoline 18
in 5 mL of anhydrous THF under argon, was added 41.4 mg of
(10% wt) Pd/C (0.04 mmol) in one portion. The reaction flask
was evacuated and purged with H2 gas four times before allow-
ing the reaction to proceed at room temperature for 6 h, at which
time it was judged complete by TLC analysis, oxazoline acid
Rf = 0.10 (EtOAc). The mixture was purged with argon, filtered
through a Whatman 0.2 μm, Puradisc 25 PP filter, attached to a
10 mL syringe. The filtered catalyst was rinsed with THF
(8 mL), followed by CHCl3 (8 mL), and the combined solution
was concentrated to afford 20 in quantitative yield, as a light
yellow-colored solid: 1H NMR (600 MHz, DMSO-d6) δ 7.25 (d,
J = 8.5 Hz, 1H), 6.11 (dd, J = 8.5, 2.1 Hz, 1H), 6.05 (d, J = 2.1 Hz,
1H), 5.88 (br. s., 2H), 4.85 (dd, J = 10.0, 7.0, 1H), 4.54–4.45 (m,
2H); 13C NMR (150 MHz, DMSO-d6) δ 172.3, 166.6, 160.9,
154.4, 128.9, 106.0, 98.9, 97.7, 68.7, 66.4; HRMS (ESI) m/z
+
[M + H]+: calcd for C10H11N2O4 , 223.0713; found, 223.0730.
N-Boc-Protected oxazoline acid, 21
+
m/z [M + Na]+: calcd for C17H24N2NaO4 , 343.1628; found,
+
343.1619. HRMS (ESI) m/z [M + H]+: calcd for C17H25N2O4 ,
To a solution of 0.643 g (1.28 mmol) of N-Boc oxazoline 19 in
28 mL of anhydrous THF under argon, was added 0.257 g of
(10% wt) Pd/C (0.24 mmol) in one portion. The system was
evacuated and purged with H2 gas four times before allowing the
reaction to proceed at room temperature for 21 h, when it was
judged complete by TLC analysis, oxazoline acid Rf = 0.10
(1 : 1, EtOAc/hexanes). The mixture was evacuated and purged
with argon, then filtered through a celite pad with 70 mL of
THF. The filtrate was concentrated to afford 21 in quantitative
yield, as a brown solid with the appearance of a dried film which
was used without further purification: 1H NMR (600 MHz,
DMSO-d6) δ 9.68 (s, 1H), 7.49 (d, J = 8.5 Hz, 1H), 7.17 (d, J =
1.8 Hz, 1H), 7.04 (dd, J = 8.7, 1.9 Hz, 1H), 4.93 (dd, J = 10.0,
7.3 Hz, 1H), 4.62–4.52 (m, 2H), 1.48 (s, 9H); 13C NMR
(150 MHz, DMSO-d6) δ 172.0, 165.9, 159.9, 152.4, 144.8, 128.5,
109.1, 104.5, 103.8, 79.7, 69.2, 66.8, 28.0; HRMS (ESI) m/z
321.1809; found, 321.1801.
N-Cbz-Protected acid, 28
To a solution of 0.115 g (0.18 mmol) of N-Cbz-protected methyl
ester 27,23 in 4 mL of THF, was added a premixed solution of
14.5 mg (0.60 mmol) of anhydrous LiOH in 4 mL of H2O and
the mixture was allowed to stir at room temperature. After 2 h,
the reaction was judged complete by TLC analysis, carboxylic
acid Rf = 0.25 (EtOAc, CAM stain). The solution was acidified
to pH < 2 (pH paper) using 1 M HCl and partitioned between
20 mL of EtOAc and 20 mL of H2O. The aqueous layer was
extracted with EtOAc (3 × 15 mL). The organic extracts were
combined, washed with brine (2 × 25 mL), dried over Na2SO4,
filtered and concentrated to afford 28 in quantitative yield, as a
colorless oil: 1H NMR (600 MHz, CDCl3) δ 7.42–7.28 (m,
10H), 5.60 (d, J = 7.9 Hz, 1H), 5.16–5.07 (m, 2H), 4.83–4.76
(m, 2H), 4.40–4.33 (m, 1H), 3.77–3.54 (m, 2H), 2.38 (t, J =
7.5 Hz, 2H), 1.94–1.19 (m, 32H), 0.89 (t, J = 7.0 Hz, 3H);
13C NMR (150 MHz, CDCl3) δ 175.9, 175.4, 156.5, 136.5,
134.5, 129.3, 129.2, 129.0, 128.7, 128.3, 128.2, 76.6, 67.2,
53.8, 44.8, 32.5, 32.1, 31.7, 29.9, 29.9, 29.9, 29.9, 29.8, 29.6,
29.6, 29.6, 26.5, 24.8, 22.9, 22.2, 14.3; HRMS (ESI) m/z
+
[M + H]+: calcd for C15H19N2O6 , 323.1238; found, 323.1250.
(R)-3-Hydroxybutanoic acid, 2526
A solution of 0.453 g (3.56 mmol) of sodium (R)-3-hydroxy-
butanoate 24 in 4 mL of H2O was acidified with 1 M HCl by
dropwise addition until pH = 1 (pH paper). The mixture was par-
titioned between 15 mL of (5 : 95, isopropyl alcohol/EtOAc) and
20 mL of H2O. The aqueous layer was extracted with 5 : 95, iso-
propyl alcohol/EtOAc (5 × 15 mL). The organic extracts were
combined, washed with brine (1 × 25 mL), dried over Na2SO4,
filtered and concentrated to afford 0.256 g (69%) of 25 as a col-
+
[M + H]+: calcd for C37H57N2O6 , 625.4211; found, 625.4205.
N-Cbz-Protected mycobactin T fragment, 29
1
orless oil: H NMR (600 MHz, CDCl3) δ 4.27–4.21 (m, 1H),
To a solution of 0.420 g (0.67 mmol) of combined lots of 28
and 0.202 g (0.63 mmol) of cobactin T, 26, in 3 mL of an-
hydrous CH2Cl2 under argon, was added 78.8 mg (0.65 mmol)
of DMAP in one portion, followed by 0.640 g (3.34 mmol) of
EDC·HCl. The reaction was stirred at room temperature for 24 h
and monitored by TLC, mycobactin fragment Rf = 0.47 (EtOAc,
CAM stain). Since it was observed that a significant amount of
26 was still present, 0.236 g (1.23 mmol) of EDC·HCl was
2.56–2.46 (m, 2H), 1.25 (d, J = 6.5 Hz, 3H); 13C NMR
(150 MHz, CDCl3) δ 177.5, 64.6, 42.7, 22.5.
Cobactin T, 2627
To a suspension of 0.145 g (1.39 mmol) of (R)-3-hydroxybuta-
noic acid 25, a solution of 0.395 g (1.25 mmol) of O-Bn-
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This journal is © The Royal Society of Chemistry 2012

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added in one portion and the mixture was stirred at room temp-
erature under argon for an additional 24 h. The crude mixture
was partitioned between 50 mL of EtOAc and 50 mL of H2O.
The aqueous layer was extracted with EtOAc (3 × 20 mL). The
organic extracts were combined, washed with brine (2 × 40 mL),
dried over Na2SO4, filtered, concentrated and purified by silica
gel chromatography with EtOAc as eluent to afford 0.514 g
(88%) of 29 as a colorless, opaque oil: 1H NMR (600 MHz,
CDCl3) δ 7.46–7.28 (m, 15H), 7.01 (d, J = 6.2 Hz, 1H), 5.64 (d,
J = 8.2 Hz, 1H), 5.33 (td, J = 12.1, 6.0 Hz, 1H), 5.13–5.03 (m,
2H), 4.98–4.94 (m, 1H), 4.89–4.83 (m, 1H), 4.79 (s, 2H), 4.45
(ddd, J = 11.3, 6.5, 1.9 Hz, 1H), 4.32 (td, J = 7.7, 5.4 Hz, 1H),
3.68–3.43 (m, 4H), 2.56–2.44 (m, 2H), 2.37 (t, J = 7.3 Hz, 2H),
1.99–1.20 (m, 41H), 0.89 (t, J = 7.0 Hz, 3H); 13C NMR
(150 MHz, CDCl3) δ 171.6 170.3, 168.4, 156.3, 136.6, 135.2,
129.9, 129.8, 129.3, 129.1, 128.9, 128.8, 128.7, 128.7, 128.3,
128.3, 128.3, 77.0, 76.6, 69.3, 67.0, 54.2, 52.9, 51.9, 51.9, 42.8,
32.6, 32.1, 32.1, 31.7, 29.9, 29.9, 29.9, 29.8, 29.7, 29.6, 29.6,
27.8, 27.7, 26.7, 26.4, 24.8, 22.9, 22.6, 19.8, 14.3; HRMS (ESI)
(150 MHz, CDCl3) δ 171.1, 170.9, 170.3, 168.4, 168.1, 161.8,
152.4, 135.3, 130.2, 129.8, 129.3, 129.1, 129.1, 128.9, 128.8,
106.8, 101.3, 101.0, 77.0, 76.5, 69.7, 69.6, 68.0, 52.9, 52.4,
51.9, 42.6, 32.6, 32.1, 32.1, 31.6, 29.9, 29.9, 29.9, 29.8, 29.7,
29.7, 29.6, 27.8, 26.7, 26.4, 24.8, 22.9, 22.7, 20.0, 14.4; HRMS
(ESI) m/z [M + H]+: calcd for C56H81N6O10+, 997.6009; found,
997.6006.
N-Boc, O-Benzyl-protected mycobactin T, 32
A solution of 95.8 mg (0.11 mmol) of 30 in 5 mL of anhydrous
CH2Cl2 under argon was cooled to 0 °C (ice-H2O bath) and
stirred for 15 min. Then 0.028 mL (0.16 mmol) of anhydrous
DIPEA was added dropwise. To the stirring mixture, a premixed
solution of 35.0 mg (0.11 mmol) of N-Boc oxazoline acid 21 in
1 mL of anhydrous THF was added slowly, followed by 15.4 mg
(0.11 mmol) of HOBt and 21.0 mg (0.11 mmol) of EDC·HCl in
one portion. The homogeneous solution was stirred at room
temperature under argon for 48 h and monitored by TLC, myco-
bactin 32 Rf = 0.38 (EtOAc, CAM stain). Since it was deter-
mined that a significant amount of the of intermediate 30
was still present, the mixture was cooled to 0 °C, and 0.01 mL
(0.06 mmol) of anhydrous DIPEA was added, followed by
18.0 mg (0.06 mmol) of N-Boc oxazoline acid 21, and 10.0 mg
(0.05 mmol) of EDC·HCl in one portion. The homogeneous
solution was stirred at room temperature, under argon for 24 h
and was then partitioned between 25 mL of EtOAc and 30 mL
of H2O. The aqueous layer was extracted with EtOAc (3 ×
15 mL). The organic extracts were combined, washed with brine
(2 × 40 mL), dried over Na2SO4, filtered, concentrated and
purified by silica gel chromatography with EtOAc as the eluent
to afford 62.3 mg (52%) of 32, as a colorless, opaque residue:
1H NMR (600 MHz, CDCl3) δ 11.49 (br. s., 1H), 7.58 (d, J =
8.5 Hz, 1H), 7.43–7.32 (m, 10H), 7.05–6.97 (m, 4H), 6.69 (s,
1H), 5.39–5.32 (m, 1H), 4.98 (d, J = 10.6 Hz, 1H), 4.93–4.87
(m, 2H), 4.75 (s, 2H), 4.62–4.56 (m, 2H), 4.53 (td, J = 7.6,
5.6 Hz, 1H), 4.46 (ddd, J = 11.3, 6.5, 1.9 Hz, 1H), 3.67–3.52
(m, 3H), 3.48 (dd, J = 15.8, 5.0 Hz, 1H), 2.58 (dd, J = 14.8,
7.2 Hz, 1H), 2.49 (dd, J = 14.7, 5.3 Hz, 1H), 2.33 (t, J = 7.5 Hz,
2H), 2.00–1.21 (m, 50H), 0.88 (t, J = 7.0 Hz, 3H); 13C NMR
(150 MHz, CDCl3) δ 171.0, 170.7, 170.3, 168.4, 167.6, 161.0,
152.2, 144.1, 135.3, 135.2, 129.8, 129.8, 129.6, 129.3, 129.1,
129.0, 128.9, 128.8, 109.3, 105.5, 105.1, 81.3, 76.5, 69.6, 69.6,
68.1, 52.9, 52.4, 51.9, 42.6, 32.6, 32.1, 31.9, 31.6, 29.9, 29.9,
29.9, 29.7, 29.7, 29.6, 29.6, 29.6, 28.5, 27.7, 26.6, 26.4, 24.8,
22.9, 22.7, 20.0, 14.3; HRMS (ESI) m/z [M + H]+: calcd for
C61H89N6O12+, 1097.6533; found, 1097.6516.
m/z [M + Na]+: calcd for C54H78N4NaO9 , 949.5661; found,
+
949.5641. HRMS (ESI) m/z [M + H]+: calcd for C54H79N4O9 ,
+
927.5842; found, 927.5833.
4-Amino, O-benzyl-protected mycobactin T, 31
Intermediate deprotection: A solution of 45.1 mg (0.05 mmol)
of N-Cbz-protected mycobactin T fragment 29, in 5.5 mL of
anhydrous CH2Cl2 under argon, was cooled to 0 °C (ice-H2O
bath) for 15 min. Then 1.5 mL of HBr/CH3CO2H (33% wt)
were added slowly and, after 15 min, the yellow-colored solution
was allowed to warm to room temperature. After 1.5 h, the reac-
tion was judged complete by TLC, HBr salt Rf = 0.02 (EtOAc,
CAM stain). The crude mixture was concentrated, co-evaporated
with CHCl3 (2 × 5 mL), toluene (2 × 5 mL), and CHCl3 (2 ×
5 mL) to afford 35.4 mg (83%) of the corresponding HBr salt 30
as an orange residue that was used without further characteri-
zation. Mycobactin assembly: To a mixture of 39.3 mg
(0.04 mmol) of intermediate 30, 15.0 mg (0.07 mmol) of
4-amino oxazoline acid 20, and 11.6 mg (0.09 mmol) of HOBt
in 3 mL of anhydrous CH2Cl2 under argon and 0.013 mL
(0.07 mmol) of anhydrous DIPEA were added dropwise,
followed by 19.2 mg (0.10 mmol) of EDC·HCl in one portion.
The homogeneous solution was stirred at room temperature
under argon for 18 h and monitored by TLC, mycobactin 31
Rf = 0.30 (EtOAc, CAM stain). The crude mixture was parti-
tioned between 25 mL of EtOAc and 30 mL of H2O. The
aqueous layer was extracted with EtOAc (3 × 15 mL). The
organic extracts were combined, washed with brine (2 × 40 mL),
dried over Na2SO4, filtered, concentrated and purified by silica
gel chromatography with EtOAc as eluent to afford 19.0 mg
Acetylated 4-amino mycobactin T, 34
Mycobactin deprotection: To remove traces of iron, all the glass-
ware involved was washed with 6 M HCl, rinsed thoroughly
with deionized H2O, until pH > 6 (pH paper), washed with
acetone and oven-dried. To a mixture of 7.7 mg (0.008 mmol) of
31 in 5 mL of anhydrous THF under argon, was added 3.2 mg
of (10% wt) Pd/C (0.003 mmol) in one portion. The reaction
flask was evacuated and purged with H2 gas four times before
allowing the reaction to proceed at room temperature for 24 h.
The mixture was evacuated and purged with argon, then filtered
1
(42%) of 31 as a colorless oil: H NMR (600 MHz, CDCl3) δ
11.55 (br. s., 1H), 7.48–7.33 (m, 11H), 7.00 (dd, J = 11.0,
7.2 Hz, 2H), 6.26 (d, J = 2.3 Hz, 1H), 6.18 (dd, J = 8.4, 2.2 Hz,
1H), 5.39–5.33 (m, 1H), 4.99 (d, J = 10.3 Hz, 1H), 4.91–4.83
(m, 2H), 4.76 (s, 2H), 4.61–4.49 (m, 3H), 4.46 (ddd, J = 11.2,
6.5, 1.9 Hz, 1H), 4.05 (s, 2H), 3.66–3.45 (m, 4H), 2.59 (dd, J =
14.7, 7.3 Hz, 1H), 2.52–2.47 (m, 1H), 2.33 (t, J = 7.3 Hz, 2H),
1.96–1.19 (m, 41H), 0.89 (t, J = 7.0 Hz, 3H); 13C NMR
This journal is © The Royal Society of Chemistry 2012
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through a Whatman 0.2 μm, Puradisc 25 PP filter, attached to a
10 mL syringe. The filtered catalyst was rinsed with CHCl3 (3 ×
10 mL), and the combined crude filtrate was concentrated to
afford mycobactin 33 as a white, opaque residue that was used
without further characterization. Acetylation: To a suspension of
the above intermediate 33 in 3 mL of anhydrous CH3CN under
argon, was added 0.13 mL (1.2 mmol) of distilled acetic an-
hydride dropwise. The resulting suspension was allowed to stir
under argon for 50 h. The reaction was monitored by LCMS and
after 24 h, a mixture of tri and tetra-acetylated product was
observed. The crude mixture was then concentrated, co-evapo-
rated with CHCl3 (5 × 3 mL) and purified over iron-free silica
gel35 with 1 : 9, isopropyl alcohol/EtOAc as eluent, to afford
5.3 mg (70% over two steps) of the acetylated mycobactin 34 as
a light, pink-colored residue: 1H NMR (600 MHz, CDCl3) δ
7.93 (d, J = 8.5 Hz, 1H), 7.59–7.51 (m, 2H), 7.33 (d, J =
7.9 Hz, 1H), 7.03 (d, J = 7.9 Hz, 1H), 6.93 (d, J = 6.5 Hz, 1H),
5.35 (dq, J = 12.5, 6.3 Hz, 1H), 4.85 (dd, J = 10.9, 7.6 Hz, 1H),
4.60 (dd, J = 10.4, 6.3 Hz, 1H), 4.54 (dd, J = 10.9, 8.8 Hz, 1H),
4.51–4.46 (m, 2H), 3.97 (dd, J = 16.1, 12.0 Hz, 1H), 3.71–3.50
(m, 3H), 2.58 (dd, J = 14.7, 6.7 Hz, 1H), 2.48 (dd, J = 14.7,
5.6 Hz, 1H), 2.41 (s, 3H), 2.25–2.12 (m, 9H), 2.02 (m, 2H),
1.86–1.20 (m, 41H), 0.89 (t, J = 7.0 Hz, 3H); 13C NMR
(150 MHz, CDCl3) δ 171.9, 171.2, 170.5, 168.6, 168.4, 167.7,
163.0, 151.1, 142.5, 131.8, 116.4, 115.3, 114.4, 69.8, 69.6, 69.5,
53.5, 52.1, 42.7, 32.1, 32.1, 31.6, 29.9, 29.9, 29.9, 29.9, 29.7,
4.54 (dd, J = 11.0, 8.7 Hz, 1H), 4.51–4.43 (m, 2H), 3.97 (dd,
J = 16.1, 12.3 Hz, 1H), 3.69–3.52 (m, 3H), 2.58 (dd, J = 14.7,
7.0 Hz, 1H), 2.48 (dd, J = 14.7, 5.6 Hz, 1H), 2.40 (s, 3H), 2.21
(s, 3H), 2.15 (s, 3H), 2.07–1.97 (m, 2H), 1.87–1.20 (m, 50H),
0.88 (t, J = 7.0 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 172.0,
171.3, 170.5, 168.4, 167.7, 163.1, 152.1, 151.3, 143.2, 131.8,
115.2, 114.1, 113.0, 81.8, 69.7, 69.6, 69.4, 53.5, 52.1, 52.1,
42.7, 32.1, 32.1, 31.6, 29.9, 29.9, 29.9, 29.9, 29.9, 29.7, 29.6,
29.6, 29.5, 28.5, 28.4, 27.9, 26.3, 22.9, 22.6, 21.7, 19.9, 18.7,
+
18.3, 14.4; HRMS (ESI) m/z [M + H]+: calcd for C53H83N6O15
,
1043.5911; found, 1043.5918.
N-Maleimide acetylated mycobactin T derivative, 40
N-Boc Deprotection: To a solution of 10.0 mg (0.01 mmol) of
intermediate 36 in 3 mL of anhydrous CH2Cl2 under argon, was
added 1 mL of TFA slowly. After 5 min, the argon inlet was
removed and the reaction was allowed to proceed at room temp-
erature for 2 h until judged complete by TLC, 37 Rf = 0.28
(1 : 9, isopropyl alcohol/EtOAc). The crude mixture was concen-
trated and co-evaporated with CHCl3 (5 × 3 mL) to afford a
yellow residue that was used without further characterization.
Acid chloride preparation: To
a solution of 32.5 mg
(0.16 mmol) of maleimide-containing acid 38,28 in 3 mL of
anhydrous CH2Cl2 under argon, was added 0.032 mL
(0.37 mmol) of oxalyl chloride, (COCl)2 slowly followed by
0.002 mL (0.03 mmol) of anhydrous DMF. The reaction was
allowed to proceed for 4 h until judged complete by TLC, 39
Rf = 0.21 (1 : 1, EtOAc/hexanes, KMnO4 stain), the mixture was
then concentrated, co-evaporated with toluene (3 × 5 mL), and
CHCl3 (3 × 5 mL) to afford a yellow residue that was used
immediately without further characterization. Acylation: A solu-
tion of (0.01 mmol) of intermediate 37 in 2 mL of anhydrous
CH2Cl2 under argon, was cooled to −78 °C (dry-ice/acetone
bath) and stirred for 15 min. Then 0.06 mL of anhydrous DIPEA
(0.34 mmol) was added dropwise, followed by (0.16 mmol) of a
premixed solution of intermediate 39 in 1.5 mL of anhydrous
CH2Cl2. After 30 min, the mixture was allowed to warm to room
temperature where it was stirred under argon for 16 h. The crude
reaction mixture was then partitioned between EtOAc (20 mL)
and H2O (20 mL). The organic layer was washed with brine (4 ×
15 mL), dried over Na2SO4, filtered, concentrated and the
residue was purified by iron-free silica gel35 chromatography,
TLC, 40 Rf = 0.11 (EtOAc, CAM stain, plate developed twice)
with 1 : 9, isopropyl alcohol/EtOAc as the eluent to afford
9.6 mg (89%) of a light-pink oil: 1H NMR (600 MHz, CDCl3) δ
7.92 (d, J = 8.8 Hz, 1H), 7.78 (br. s., 1H), 7.60 (br. s., 1H), 7.38
(d, J = 8.2 Hz, 1H), 7.04 (d, J = 7.9 Hz, 1H), 6.96 (d, J =
6.5 Hz, 1H), 6.71 (d, J = 15.8 Hz, 2H), 5.34 (dq, J = 12.5,
6.4 Hz, 1H), 4.85 (dd, J = 11.0, 7.5 Hz, 1H), 4.60 (dd, J = 10.9,
5.9 Hz, 1H), 4.54 (dd, J = 11.0, 8.7 Hz, 1H), 4.51–4.45 (m, 2H),
3.97 (dd, J = 15.8, 11.7 Hz, 1H), 3.69–3.52 (m, 5H), 2.58 (dd,
J = 14.7, 6.7 Hz, 1H), 2.48 (dd, J = 14.7, 5.6 Hz, 1H),
2.45–2.36 (m, 5H), 2.35–2.33 (m, 1H), 2.25–2.11 (m, 7H), 2.02
(m, 2H), 1.85–1.19 (m, 43H), 0.88 (t, J = 7.0 Hz, 3H);
13C NMR (150 MHz, CDCl3) δ 176.6, 171.9, 171.3, 171.2,
171.2, 171.2, 171.0, 170.5, 168.5, 167.7, 163.1, 151.1, 142.7,
134.4, 134.4, 134.3, 131.8, 116.4, 115.1, 114.4, 69.8, 69.5, 69.5,
29.6, 29.6, 29.5, 27.9, 26.3, 25.0, 22.9, 22.6, 21.7, 19.9, 18.7,
18.3, 14.4; HRMS (ESI) m/z [M + H]+: calcd for C50H77N6O14
,
+
985.5492; found, 985.5502.
N-Boc-Protected, acetylated mycobactin T, 36
Mycobactin deprotection: To remove traces of iron, all the glass-
ware involved was washed with 6 M HCl, rinsed thoroughly
with deionized H2O, until pH > 6 (pH paper), washed with
acetone and oven-dried. To a mixture of 62.3 mg (0.056 mmol)
of 32 in 11 mL of anhydrous THF under argon, was added
36.2 mg of (10% wt) Pd/C (0.034 mmol) in one portion. The
reaction flask was evacuated and purged with H2 gas four times
before allowing the reaction to proceed at room temperature for
30 h. The reaction flask was evacuated and purged with argon.
The reaction mixture was filtered through a Whatman 0.2 μm,
Puradisc 25 PP filter, attached to a 10 mL syringe. The filtered
catalyst was rinsed with CHCl3 (3 × 10 mL), and the combined
solution was concentrated to afford mycobactin 35 as a light,
tan-colored residue that was used without further characteri-
zation. Acetylation: To a solution of 35 in 1 mL of anhydrous
THF under argon, was added 1 mL of anhydrous CH3CN,
followed by dropwise addition of 1 mL (10.6 mmol) of distilled
acetic anhydride. The homogeneous solution was stirred for 48 h
under argon. The reaction mixture was then concentrated, co-
evaporated with CHCl3 (8 × 3 mL) and purified over iron-free
silica gel35 with 2 : 8, CH2Cl2/EtOAc as the eluent to afford
51.5 mg (87%) of 36 as a light-pink residue: 1H NMR
(600 MHz, CDCl3) δ 7.89 (d, J = 8.8 Hz, 1H), 7.44 (br. s., 1H),
7.15 (dd, J = 8.8, 2.1 Hz, 1H), 7.03 (d, J = 7.9 Hz, 1H), 6.94 (d,
J = 6.5 Hz, 1H), 6.73 (s, 1H), 5.35 (dq, J = 12.5, 6.3 Hz, 1H),
4.84 (dd, J = 11.0, 7.5 Hz, 1H), 4.60 (dd, J = 10.3, 6.5 Hz, 1H),
7592 | Org. Biomol. Chem., 2012, 10, 7584–7593
This journal is © The Royal Society of Chemistry 2012

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Acknowledgements
We acknowledge the University of Notre Dame and the NIH (AI
054193) for supporting this work. The excellent assistance of
Sang Hyun Cho with anti-TB assays, performed at the Institute
for Tuberculosis Research, UIC. We gratefully acknowledge the
use of the NMR facilities provided by the Lizzadro Magnetic
Resonance Research Center at The University of Notre Dame
(UND) and the mass spectrometry services provided by The
UND Mass Spectrometry & Proteomics Facility (Mrs N. Sevova,
Dr W. Boggess, and Dr M. V. Joyce; supported by the National
Science Foundation under CHE-0741793). We thank Mrs Patri-
cia A. Miller (UND) for antibacterial susceptibility testing.
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This journal is © The Royal Society of Chemistry 2012
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